Current approaches to treating disease by administering therapeutic proteins include in vitro production of therapeutic proteins for conventional pharmaceutical delivery (e.g. intravenous, subcutaneous, or intramuscular injection) and, more recently, gene therapy.
Proteins of therapeutic interest can be produced by introducing exogenous DNA encoding the protein of therapeutic interest into appropriate cells. For example, a vector which includes exogenous DNA encoding a therapeutic protein can be introduced into cells and the encoded protein expressed. It has also been suggested that endogenous cellular genes and their expression may be modified by gene targeting. See for example, U.S. Pat. Nos. 5,272,071, 5,641,670, WO 91/06666, WO 91/06667 and WO 90/11354.
The invention is based, in part, on the use of homologous recombination between a double stranded DNA sequence and a selected target DNA, e.g., chromosomal DNA in a cell, promoted by providing an agent which enhances homologous recombination, e.g., Rad52, and an agent which inhibits non-homologous end joining, e.g., a Ku inactivating agent, in sufficiently close proximity to the DNA sequence at the targeted site. It is predicted that a higher rate of homologous recombination occurred in the presence of both Rad52 and a Ku inactivating agent than in their absence. In addition, it is predicted that gene targeting aimed at altering a targeted site in a DNA, e.g., a targeted site in the chromosomal DNA in a cell, using a selected DNA sequence as a template can be promoted by providing a Rad52 protein and a Ku inactivating agent, e.g., an anti-Ku antibody. By providing a Rad52 protein and a Ku inactivating agent in close proximity to the selected DNA sequence and the target site, a higher rate of alteration by gene targeting occurs than in the absence of a Rad52 protein and a Ku inactivating agent, e.g., an anti-Ku antibody.
Accordingly, in one aspect, the invention features, a method of promoting an alteration at a selected site in a target DNA, e.g., in the chromosomal DNA of a cell. The method includes providing, at the site: (a) a double stranded DNA sequence which includes a selected DNA sequence; (b) an agent which enhances homologous recombination, e.g., a Rad52 protein or a functional fragment thereof, or a DNA sequence which encodes Rad52 or a functional fragment thereof; and (c) an agent which inhibits non-homologous end joining, e.g., an agent which inactivates Ku, and allowing the alteration to occur. In a preferred embodiment, components (a), (b), and (c) are provided, e.g., introduced into the cell, such that, at the site of an interaction between the selected DNA sequence and the target DNA, the concentration of the agent which enhances homologous recombination and of the agent which inhibits non-homologous end joining are sufficient that an alteration of the site, e.g., homologous recombination or gene correction between the selected DNA sequence and the target DNA, occurs at a higher rate than would occur in the absence of the supplied agent which enhances homologous recombination and the agent which inhibits non-homologous end joining. The agent which inhibits non-homologous end joining is preferably provided locally. Preferably the agent which inhibits non-homologous end joining is a Ku inactivating agent such as an anti-Ku antibody.
Components (a), (b), and (c) can be introduced together, which is preferred, or separately. In addition, two of the components can be introduced together and the third can be introduced separately. For example, the DNA sequence and the agent which enhances homologous recombination, e.g., Rad52, can be introduced together or the DNA sequence and the agent which inhibits non-homologous end joining, e.g., a Ku inactivating agent, can be introduced together. In another preferred embodiment, the agent which enhances homologous recombination and the agent which inhibits non-homologous end joining can be introduced together.
Two, or preferably all, of the components can be provided as a complex. In a preferred embodiment, the method includes contacting the target DNA, e.g., by introducing into the cell, a complex which includes: (a) a double stranded DNA sequence which includes the selected DNA sequence; (b) an agent which enhances homologous recombination, e.g., a Rad52 protein or functional fragment thereof; and (c) an agent which inhibits non-homologous end joining, e.g., a Ku inactivating agent such as an anti-Ku antibody or a Ku-binding oligomer or polymer.
In a preferred embodiment, one, or more, preferably all of the components are provided by local delivery, e.g., microinjection, and are not expressed from the target genome or another nucleic acid. In a particularly preferred embodiment, the agent which inhibits non-homologous end joining, e.g., a Ku inhibiting agent, is provided by local delivery, e.g., microinjection, and is not expressed from the target genome or another nucleic acid.
In a preferred embodiment, the agent which inhibits non-homologous end joining is: an agent which inactivates hMre11, e.g., an anti-hMre11 antibody or a hMre11-binding oligomer or polymer; an agent which inactivates hRad50, e.g., an anti-hRad50 antibody or a hRad50-binding oligomer or polymer; an agent which inactivates Nbs1, e.g., an anti-Nbs1 antibody or a hNbs1-binding oligomer or polymer; an agent which inactivates human ligase 4 (hLig4), e.g., an anti-hLig4 antibody or a hLig4-binding oligomer or polymer; an agent which inactivates hXrcc4, e.g., an anti-hXrcc4 antibody or a hXrcc4-binding oligomer or polymer; an agent which inactivates a human homolog of Rap1, e.g., an antibody to a human homolog of Rap1 or an oligomer or polymer which binds a human homolog of Rap1; an agent which inactivates a human homolog of Sir2304, e.g., an antibody to a human homolog of Sir2304 or an oligomer or polymer which binds a human homolog of Sir2304; an agent which inactivates Ku, e.g., an anti-Ku antibody or a Ku-binding oligomer or polymer. Any of the agents which inhibit non-homologous end joining can be administered alone or can be administered in combination with one or more of the other agents which inhibit non-homologous end joining.
In a preferred embodiment, the DNA sequence is a linear DNA sequence. In a preferred embodiment, the linear DNA sequence can have one or more single stranded overhang(s).
In a preferred embodiment, the selected DNA sequence is flanked by a targeting sequence. The targeting sequence is homologous to the target, e.g., homologous to DNA adjacent to the site where the target DNA is to be altered or to the site where the selected DNA sequence is to be integrated. Such flanking sequence can be present at one or more, preferably both ends of the selected DNA sequence. If two flanking sequences are present, one should be homologous with a first region of the target and the other should be homologous to a second region of the target.
In a preferred embodiment, the DNA sequence has one or more protruding single stranded end, e.g., one or both of the protruding ends are 3xe2x80x2 ends or 5xe2x80x2 ends.
In a preferred embodiment, the agent which enhances homologous recombination is: a Rad52 protein or a functional fragment thereof; a Rad51 protein or a functional fragment thereof; a Rad54 protein or a functional fragment thereof; or a combination thereof.
In a preferred embodiment, the agent which enhances homologous recombination is adhered to, e.g., coated on, the DNA sequence. In a preferred embodiment, the Rad52 protein or functional fragment thereof is adhered to, e.g., coated on, the DNA sequence.
In a preferred embodiment, the Rad52 protein or fragment thereof is human Rad52 (hRad52).
In a preferred embodiment, the anti-Ku antibody is: an anti-Ku70 antibody; an anti-Ku80 antibody. In a preferred embodiment, the anti-Ku antibody is: a humanized antibody; a human antibody; an antibody fragment, e.g., a Fab, Fabxe2x80x2, F(abxe2x80x2)2 or F(v) fragment.
In a preferred embodiment, at least one anti-Ku antibody is covalently linked to: the DNA sequence; the Rad52 protein or fragment thereof. In another preferred embodiment, at least one anti-Ku antibody is non-covalently linked to: the DNA sequence; the Rad52 protein or fragment thereof.
In a preferred embodiment, an anti-Ku70 antibody and an anti-Ku80 antibody is provided, e.g., as components of a complex.
In a preferred embodiment, the cell is: a eukaryotic cell. In a preferred embodiment, the cell is of fungal, plant or animal origin, e.g., vertebrate origin. In a preferred embodiment, the cell is: a mammalian cell, e.g., a primary or secondary mammalian cell, e.g., a fibroblast, a hematopoietic stem cell, a myoblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a cell comprising a formed element of the blood, a muscle cell and precursors of these somatic cells; a transformed or immortalized cell line. Preferably, the cell is a human cell. Examples of immortalized human cell line useful in the present method include, but are not limited to: a Bowes Melanoma cell (ATCC Accession No. CRL 9607), a Daudi cell (ATCC Accession No. CCL 213), a HeLa cell and a derivative of a HeLa cell (ATCC Accession Nos. CCL2 CCL2.1, and CCL 2.2), a HL-60 cell (ATCC Accession No. CCL 240), a HT1080 cell (ATCC Accession No. CCL 121), a Jurkat cell (ATCC Accession No. TIB 152), a KB carcinoma cell (ATCC Accession No. CCL 17), a K-562 leukemia cell (ATCC Accession No. CCL 243), a MCF-7 breast cancer cell (ATCC Accession No. BTH 22), a MOLT-4 cell (ATCC Accession No. 1582), a Namalwa cell (ATCC Accession No. CRL 1432), a Rafji cell (ATCC Accession No. CCL 86), a RPMI 8226 cell (ATCC Accession No. CCL 155), a U-937 cell (ATCC Accession No. 1593), WI-28VA13 sub line 2R4 cells (ATCC Accession No. CLL 155), a CCRF-CEM cell (ATCC Accession No. CCL 119) and a 2780AD ovarian carcinoma cell (Van Der Blick et al., Cancer Res. 48:5927-5932, 1988), as well as heterohybridoma cells produced by fusion of human cells and cells of another species. In another embodiment, the immortalized cell line can be cell line other than a human cell line, e.g., a CHO cell line, a COS cell line.
In a preferred embodiment, the components, e.g., the components of a complex, are introduced into the cell by microinjection.
In one preferred embodiment, the selected DNA sequence differs from the target DNA by less than 10, 8, 6, 5, 4, 3, 2, or by a single nucleotide, e.g., by a substitution, or a deletion, or an insertion.
In a preferred embodiment, the target DNA includes a mutation, e.g., the target sequence differs from wild-type sequence by about 10, 8, 6, 5, 4, 3, 2 or by a single nucleotide. Preferably, the mutation is a point mutation, e.g., a mutation due to an insertion, deletion or a substitution.
In a preferred embodiment, the target DNA includes a mutation and the mutation is associated with, e.g., causes, contributes to, conditions or controls, a disease or a dysfunction. Preferably, the disease or dysfunction is: cystic fibrosis; sickle cell anemia; hemophilia A; hemophilia B; von Willebrand disease type 3; xeroderma pigmentosa; thalassaemias; Lesch-Nylan syndrome; protein C resistance; a lysosomal storage disease, e.g., Gaucher disease, Fabry disease; mucopolysaccharidosis (MPS) type 1 (Hurley-Scheie syndrome), MPS type II (Hunter syndrome), MPS type IIIA (Sanfilio A syndrome), MPS type IIIB (Sanfilio B syndrome), MPS type IIIC (Sanfilio C syndrome), MPS type IIID (Sanfilio D syndrome), MPS type IVA (Morquio A syndrome), MPS type IVB (Morquio B syndrome), MPS type VI (Maroteaux-Larry syndrome), MPS type VII (Sly syndrome).
In a preferred embodiment, the target DNA includes a mutation and the selected DNA sequence includes a normal wild-type sequence which can correct the mutation.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the cystic fibrosis transmembrane regulator (CFTR) gene. Preferably, the mutation is one which alters the amino acid at codon 508 of the CFTR protein coding region, e.g., the mutation is a 3 base pair in-frame deletion which eliminates a phenylalanine at codon 508 of the CFTR protein. This deletion of phenylalanine-508 in the CFTR protein is found in a high percentage of subjects having cystic fibrosis. Thus, in a preferred embodiment, a selected DNA sequence including sequence encoding phenylalanine-508 as found in the wild-type CFTR gene can be used to target and correct the mutated CFTR gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the human xcex2-globin gene. Preferably, the mutation is one which alters the amino acid at the sixth codon of the xcex2-globin gene, e.g., the mutation is an A to T substitution in the sixth codon of the xcex2-globin gene. This mutation leads to a change from glutamic acid to valine in the xcex2-globin protein which is found in subjects having sickle cell anemia. Thus, in a preferred embodiment, a selected DNA which encodes a wild-type amino acid residue at codon 6, e.g., a selected DNA sequence including an A as found within the sixth codon of wild-type xcex2-globin gene, can be used to target and correct the mutated xcex2-globin gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the Factor VIII gene. For example, a mutation can be in exon 23, 24, and/or exon 25 of the Factor VIII gene. Preferably, the mutation is one which alters the amino acid at codon 2209 of the coding region of the Factor VIII protein coding region, e.g., the mutation is a G to A substitution in exon 24 of the Factor VIII gene which leads to a change from an arginine to a glutamine at amino acid 2209 of Factor VIII. Preferably, the mutation is one which alters the amino acid at codon 2229 of the coding region of the Factor VIII protein coding region, e.g., the mutation is a G to T substitution in exon 25 of the Factor VIII gene which leads to a change from a tryptophan to a cysteine at amino acid 2229 of Factor VIII. These mutations have been associated with moderate to severe hemophilia A. Thus, in a preferred embodiment, a selected DNA sequence including either DNA which encodes a wild-type amino acid at codon 2209 of the coding region of Factor VIII gene or DNA which encodes a wild-type amino acid at codon 2229 of the coding region of the Factor VIII gene, or both, can be used to target and correct the mutated Factor VIII gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the Factor IX gene. For example, in subjects having hemophilia B, most of the mutations are point mutations in the Factor IX gene. Thus, in a preferred embodiment, the selected DNA sequence can include one or more nucleotides having at least one nucleotide from the wild-type Factor IX gene, to target and correct one or more of the point mutations in the Factor IX gene associated with hemophilia B.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the von Willebrand factor gene. Preferably, the mutation is a single cytosine deletion in a stretch of 6 cytosines at positions 2679-2684 in exon 18 of the von Willebrand gene. This mutation is found in a significant percentage of subjects having von Willebrand disease type 3. Other mutations, e.g., point mutations, associated with von Willebrand disease type 3 can also be altered as described herein. Thus, in a preferred embodiment, a selected DNA sequence including sequences found in the wild-type von Willebrand gene, e.g., the six cytosines at positions 2679-2684 of the von Willebrand gene, can be used to target and correct the mutated von Willebrand gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the Xeroderma pigmentosum group G (XP-G) gene. Preferably, the mutation is a deletion of a single adenine in a stretch of three adenines at positions 19-21 of a 245 base-pair exon found in the XP-G gene. This deletion leads to xeroderma pigmentosum. Thus, in a preferred embodiment, a selected DNA including the wild-type sequence of the XP-G gene, e.g., three adenines at positions 19-21 of the 245 base-pair exon, can be used to target and correct the mutated XP-G gene.
Preferably, an agent which inactivates a mismatch repair protein such as Msh2, Msh6, Msh3, Mlh1, Pms2, Mlh3, Pms1, is also provided. The agent can be included in a complex.
In another preferred embodiment, the alteration includes homologous recombination between the selected DNA sequence and the target DNA, e.g., a chromosome.
In preferred embodiment, the selected DNA sequence differs from the target DNA by more than one nucleotide, e.g., it differs from the target by a sufficient number of nucleotides such that the target, or the selected DNA sequence has an unpaired region, e.g., a loop-out region. In such an application, Msh2, Msh6, Msh3, Mlh1, Pms2, Mlh3, Pms1 can also be provided, e.g., as part of a complex.
In a preferred embodiment, the alteration includes integration of the selected sequence into the target DNA and the selected DNA is integrated such that it is in a preselected relationship with a preselected element on the target, e.g., if one is a regulatory element and the other is a sequence which encodes a protein, the regulatory element functions to regulate expression of the protein encoding sequence. Flanking sequences which promote the selected integration can be used. The selected DNA sequence can be integrated 5xe2x80x2, 3xe2x80x2, or within, a selected target sequence, e.g., a gene or coding sequence.
In a preferred embodiment, the alteration includes integration of the selected DNA sequence and the selected DNA sequence is a regulatory sequence, e.g., an exogenous regulatory sequence. In a preferred embodiment, the regulatory sequence includes one or more of: a promoter, an enhancer, an upstream activating sequence (UAS), a scaffold-attachment region or a transcription factor-binding site. In a preferred embodiment, the regulatory sequence includes: a regulatory sequence from a metallothionein-I gene, e.g., a mouse metallothionein-I gene, a regulatory sequence from an SV-40 gene, a regulatory sequence from a cytomegalovirus gene, a regulatory sequence from a collagen gene, a regulatory sequence from an actin gene, a regulatory sequence from an immunoglobulin gene, a regulatory sequence from the HMG-CoA reductase gene, a regulatory sequence from xcex3 actin gene, a regulatory sequence from transcription activator YY1 gene, a regulatory sequence from fibronectin gene, or a regulatory sequence from the EF-1xcex1 gene.
In a preferred embodiment, the selected DNA sequence includes an exon. Preferably, the exogenous exon includes: a CAP site, the nucleotide sequence ATG, and/or encoding DNA in-frame with the targeted endogenous gene.
In a preferred embodiment, the selected DNA sequence includes a splice-donor site.
In a preferred embodiment, the selected DNA sequence includes an exogenous regulatory sequence which when integrated into the target functions to regulate an endogenous coding sequence. The selected DNA sequence can be integrated upstream of the coding region of an endogenous gene in the target or upstream of the endogenous regulatory sequence of an endogenous gene in the target. In another preferred embodiment, the selected DNA sequence can be integrated downstream of an endogenous gene or coding region or within an intron or an endogenous gene. In another preferred embodiment, the selected DNA sequence can be integrated such that the endogenous regulatory sequence of the endogenous gene is inactive, e.g., is wholly or partially deleted.
In a preferred embodiment, the selected DNA sequence is upstream of an endogenous gene and is linked to the second exon of the endogenous gene.
In a preferred embodiment, the endogenous gene encodes: a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein or a transcription factor. In a preferred embodiment, the endogenous gene encodes any of the following proteins: erythropoietin, calcitonin, growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, xcex12-interferon (IFNA2), xcex2-interferon, xcex3-interferon, nerve growth factors, FSHxcex2, TGF-xcex2, tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-xcex2, interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-granulocyte (GCSF), CSF-macrophage, CSF-granulocyte/macrophage, immunoglobulins, catalytic antibodies, protein kinase C, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNAse, xcex1-galactosidase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 antagonists, xcex1-1-antitrypsin, immune response modifiers, xcex2-glucoceramidase, xcex1-iduronidase, xcex1-L-iduronidase, glucosamine-N-sulfatase, xcex1-N-acetylglucosaminidase, acetylcoenzymeA:xcex1-glucosamine-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, xcex2-galactosidase, xcex2-glucuronidase, N-acetylgalactosamine-6-sulfatase, and soluble CD4.
In a preferred embodiment, the endogenous gene encodes follicle stimulating hormone xcex2(FSHxcex2) and the selected DNA sequence includes a regulatory sequence, e.g., a regulatory sequence which differs in sequence from the regulatory sequence of the FSHxcex2 gene. Preferably, the selected DNA sequence is flanked by a targeting sequence, e.g., such targeting sequence is present at one or more, preferably both ends of the selected DNA sequence. In a preferred embodiment, the targeting sequence is homologous to a region 5xe2x80x2 of the FSHxcex2 coding region (SEQ ID NO:1). In a preferred embodiment, the targeting sequence directs homologous recombination within the FSHxcex2 coding sequence or upstream of the FSHxcex2 coding sequence. In a preferred embodiment, the targeting sequence includes at least 20, 30, 50, 100 or 1000 contiguous nucleotides from SEQ ID NO:2, which corresponds to nucleotides xe2x88x927454 to xe2x88x921417 of human FSHxcex2 sequence (numbering is relative to the translation start site), or SEQ ID NO:3, which corresponds to nucleotides xe2x88x92696 to xe2x88x92155 of human FSHxcex2 sequence.
In a preferred embodiment, the endogenous gene encodes interferon xcex12 (IFNxcex12) and the selected DNA sequence includes a regulatory sequence, e.g., a regulatory sequence which differs in sequence from the regulatory sequence of the IFNxcex12 gene. Preferably, the selected DNA sequence is flanked by a targeting sequence, e.g., such targeting sequence is present at one or more, preferably both ends of the selected DNA sequence. In a preferred embodiment, the targeting sequence is homologous to a region 5xe2x80x2 of the IFNxcex12 coding region. In a preferred embodiment, the targeting sequence directs homologous recombination within a region upstream of the IFNxcex12 coding sequence. In a preferred embodiment, the targeting sequence includes at least 20, 30, 50, 100 or 1000 contiguous nucleotides from SEQ ID NO:4, which corresponds to nucleotides xe2x88x924074 to xe2x88x92511 of human IFNxcex12 sequence (numbering is relative to the translation start site). For example, it can include: at least 20, 30, 50, or 100 nucleotides from SEQ ID NO:7, which corresponds to nucleotides xe2x88x924074 to xe2x88x923796 of human IFNxcex12 sequence; at least 20, 30, or 50 nucleotides from SEQ ID NO:8, which corresponds to nucleotides xe2x88x92582 to xe2x88x92510 of human IFNxcex12 sequence; at least 20, 30, 50, 100, or 1000 nucleotides from SEQ ID NO:9, which corresponds to nucleotides xe2x88x923795 to xe2x88x92583 of human IFNxcex12 sequence.
In a preferred embodiment, the endogenous gene encodes granulocyte colony stimulating factor (GCSF) and the selected DNA sequence includes a regulatory sequence, e.g., a regulatory sequence which differs in sequence from the regulatory sequence of the GCSF gene. Preferably, the selected DNA sequence is flanked by a targeting sequence, e.g., such targeting sequence is present at one or more, preferably both ends of the selected DNA sequence. In a preferred embodiment, the targeting sequence is homologous to a region 5xe2x80x2 of the GCSF coding region. In a preferred embodiment, the targeting sequence directs homologous recombination within the GCSF coding sequence or upstream of the GCSF coding sequence. In a preferred embodiment, the targeting sequence includes at least 20, 30, 50, 100 or 1000 contiguous nucleotides from SEQ ID NO:5, which corresponds to nucleotides xe2x88x926,578 to 101 of human GCSF sequence (numbering is relative to the translation start site). For example, the target sequence can include 20, 30, 50, 100 or 1000 nucleotides from SEQ ID NO:6, which corresponds to nucleotides xe2x88x926,578 to xe2x88x92364 of the human GCSF gene.
In another preferred embodiment, the DNA sequence includes a coding region, e.g., the selected DNA sequence encodes a protein. In a preferred embodiment, the coding region encodes: a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein or a transcription factor. In a preferred embodiment, the coding region encodes any of the following proteins: erythropoietin, calcitonin, growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, xcex12-interferon (IFNA2), xcex2-interferon, xcex3-interferon, nerve growth factors, FSHxcex2, TGF-xcex2, tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-xcex2, interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-granulocyte (GCSF), CSF-macrophage, CSF-granulocyte/macrophage, immunoglobulins, catalytic antibodies, protein kinase C, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNAse, xcex1-galactosidase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 antagonists, xcex1-1-antitrypsin, immune response modifiers, xcex2-glucoceramidase, xcex1-iduronidase, xcex1-L-iduronidase, glucosamine-N-sulfatase, xcex1-N-acetylglucosaminidase, acetylcoenzymeA:xcex1-glucosamine-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, xcex2-galactosidase, xcex2-glucuronidase, N-acetylgalactosamine-6-sulfatase, and soluble CD4.
In a preferred embodiment, the selected DNA sequence can be integrated into the target such that it is under the control of an endogenous regulatory element. The selected DNA can be integrated downstream of an endogenous regulatory sequence or upstream of a coding region of an endogenous gene and downstream of the endogenous regulatory sequence of the gene. In another preferred embodiment, the selected DNA can be integrated downstream of an endogenous regulatory sequence such that the coding region of the endogenous gene is inactivated, e.g., is wholly or partially deleted.
In a preferred embodiment, the method further includes introducing an agent which inhibits a mismatch-repair protein, e.g., Msh2, Msh6, Msh3, Mlh1, Pms2, Mlh3, Pms1, or other mismatch repair proteins, or combinations thereof. Preferably, the agent is an agent which inhibits expression of a mismatch-repair protein, e.g., the agent is an antisense RNA. In a preferred embodiment, the agent is an antibody against a mismatch-repair protein. In a preferred embodiment, the antibody against the mismatch-repair protein is covalently or non-covalently linked to the complex.
In another aspect, the invention features, a composition, e.g., a complex of components, for promoting an alteration at a target DNA, e.g., a chromosome, e.g., a target DNA described herein, using a selected DNA sequence, e.g., a selected DNA sequence described herein, as a template. The composition includes: (a) a double stranded DNA sequence which includes a selected DNA sequence; (b) an agent which enhances homologous recombination, e.g., a Rad52 protein or a functional fragment thereof; and (c) an agent which inhibits non-homologous end joining, e.g., an agent which inactivates Ku. The composition can be used, for example, to alter the target DNA sequence by integration.
In a preferred embodiment, the agent which inhibits non-homologous end joining is: an agent which inactivates hMre11, e.g., an anti-hMre11 antibody or a hMre11-binding oligomer or polymer; an agent which inactivates hRad50, e.g., an anti-hRad50 antibody or a hRad50-binding oligomer or polymer; an agent which inactivates Nbs1, e.g., an anti-Nbs1 antibody or a hNbs1-binding oligomer or polymer; an agent which inactivates human ligase 4 (hLig4), e.g., an anti-hLig4 antibody or a hLig4-binding oligomer or polymer; an agent which inactivates hXrcc4, e.g., an anti-hXrcc4 antibody or a hXrcc4-binding oligomer or polymer; an agent which inactivates a human homolog of Rap1, e.g., an antibody to a human homolog of Rap1 or an oligomer or polymer which binds a human homolog of Rap1; an agent which inactivates a human homolog of Sir2304, e.g., an antibody to a human homolog of Sir2304 or an oligomer or polymer which binds a human homolog of Sir2304; an agent which inactivates Ku, e.g., an anti-Ku antibody or a Ku-binding oligomer or polymer. Any of the agents which inhibit non-homologous end joining can be administered alone or can be administered in combination with one or more of the other agents which inhibit non-homologous end joining.
In a preferred embodiment, the DNA sequence is a linear DNA sequence. In a preferred embodiment, the linear DNA sequence can have one or more single stranded overhang(s).
In a preferred embodiment, the selected DNA sequence is flanked by a targeting sequence. The targeting sequence is homologous to the target, e.g., homologous to DNA adjacent to the site where the target DNA is to be altered or to the site where the selected DNA sequence is to be integrated. Such flanking sequence can be present at one or more, preferably both ends of the selected DNA sequence. If two flanking sequences are present, one should be homologous to a first region of the target and the other should be homologous to a second region of the target.
In a preferred embodiment, the DNA sequence has one or more protruding single stranded end, e.g., one or both of the protruding ends are 3xe2x80x2 ends or 5xe2x80x2 ends.
In a preferred embodiment, the agent which enhances homologous recombination is: a Rad52 protein or a functional fragment thereof; a Rad51 protein or a functional fragment thereof; a Rad54 protein or a functional fragment thereof; or a combination thereof.
In a preferred embodiment, the agent which enhances homologous recombination is adhered to, e.g., coated on, the DNA sequence. In a preferred embodiment, the Rad52 protein or functional fragment thereof is adhered to, e.g., coated on, the selected DNA sequence.
In a preferred embodiment, the Rad52 protein or fragment thereof is human Rad52 (hRad52).
In a preferred embodiment, the anti-Ku antibody is: an anti-Ku70 antibody; an anti-Ku80 antibody. In a preferred embodiment, the anti-Ku antibody is: a humanized antibody; a human antibody; an antibody fragment, e.g., a Fab, Fabxe2x80x2, F(abxe2x80x2)2 or F(v) fragment.
In a preferred embodiment, at least one anti-Ku antibody is covalently linked to: the selected DNA sequence; the Rad52 protein or fragment thereof. In another preferred embodiment, at least one anti-Ku antibody is covalently linked to: the selected DNA sequence; the Rad52 protein or fragment thereof.
In a preferred embodiment, the composition includes an anti-Ku70 antibody and an anti-Ku80 antibody.
In a preferred embodiment, the selected DNA sequence differs from the target DNA by less than 10, 8, 6, 5, 4, 3, 2 or by a single nucleotide, e.g., a substitution, or a deletion, or an insertion.
In a preferred embodiment, the target DNA includes a mutation, e.g., the target sequence differs from wild-type sequence by about 10, 8, 6, 5, 4, 3, 2 or by a single nucleotide. Preferably, the mutation is a point mutation, e.g., a mutation due to an insertion, deletion or a substitution.
In a preferred embodiment, the target DNA includes a mutation and the mutation is associated with, e.g., causes, contributes to, conditions or controls, a disease or a dysfunction. Preferably, the disease or dysfunction is: cystic fibrosis; sickle cell anemia; hemophilia A; hemophilia B; von Willebrand disease type 3; xeroderma pigmentosa; thalassaemias; Lesch-Nylan syndrome; protein C resistance; a lysosomal storage disease, e.g., Gaucher disease, Fabry disease, mucopolysaccharidosis (MPS) type 1 (Hurley-Scheie syndrome), MPS type II (Hunter syndrome), MPS type IIIA (Sanfilio A syndrome), MPS type IIIB (Sanfilio B syndrome), MPS type IIIC (Sanfilio C syndrome), MPS type IIID (Sanfilio D syndrome), MPS type IVA (Morquio A syndrome), MPS type IVB (Morquio B syndrome), MPS type VI (Maroteaux-Larry syndrome), MPS type VII (Sly syndrome).
In a preferred embodiment, the target DNA includes a mutation and the selected DNA sequence includes a normal wild-type sequence which can correct the mutation.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the cystic fibrosis transmembrane regulator (CFTR) gene. Preferably, the mutation is one which alters the amino acid at codon 508 of the CFTR protein coding region, e.g., the mutation is a 3 base pair in-frame deletion which eliminates a phenylalanine at codon 508 of the CFTR protein. This deletion of phenylalanine-508 in the CFTR protein is found in a high percentage of subjects having cystic fibrosis. Thus, in a preferred embodiment, a selected DNA sequence including sequence encoding phenylalanine-508 as found in the wild-type CFTR gene can be used to target and correct the mutated CFTR gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the human xcex2-globin gene. Preferably, the mutation is one which alters the amino acid at the sixth codon of the xcex2-globin gene, e.g., the mutation is an A to T substitution in the sixth codon of the xcex2-globin gene. This mutation leads to a change from glutamic acid to valine in the xcex2-globin protein which is found in subjects having sickle cell anemia. Thus, in a preferred embodiment, a selected DNA which encodes a wild-type amino acid residue at codon 6, e.g., a selected DNA sequence including an A as found within the sixth codon of wild-type xcex2-globin gene, can be used to target and correct the mutated xcex2-globin gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the Factor VIII gene. For example, a mutation can be in exon 23, 24, and/or exon 25 of the Factor VIII gene. Preferably, the mutation is one which alters the amino acid at codon 2209 of the coding region of the Factor VIII protein coding region, e.g., the mutation is a G to A substitution in exon 24 of the Factor VIII gene which leads to a change from an arginine to a glutamine at amino acid 2209 of Factor VIII. Preferably, the mutation is one which alters the amino acid at codon 2229 of the coding region of the Factor VIII protein coding region, e.g., the mutation is a G to T substitution in exon 25 of the Factor VIII gene which leads to a change from a tryptophan to a cysteine at amino acid 2229 of Factor VIII. These mutations have been associated with moderate to severe hemophilia A. Thus, in a preferred embodiment, a selected DNA sequence including either DNA which encodes a wild-type amino acid at codon 2209 of the coding region of Factor VIII gene, or DNA which encodes a wild-type amino acid at codon 2229 of the coding region of the Factor VIII gene, or both, can be used to target and correct the mutated Factor VIII gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the Factor IX gene. For example, in subjects having hemophilia B, most of the mutations are point mutations in the Factor IX gene. Thus, in a preferred embodiment, the selected DNA sequence can include one or more nucleotides having at least one nucleotide from the wild-type Factor IX gene, to target and correct one or more of the point mutations in the Factor IX gene associated with hemophilia B.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the von Willebrand factor gene. Preferably, the mutation is a single cytosine deletion in a stretch of six cytosines at positions 2679-2684 in exon 18 of the von Willebrand gene. This mutation is found in a significant percentage of subjects having von Willebrand disease type 3. Other mutations, e.g., point mutations, associated with von Willebrand disease type 3 can also be altered as described herein. Thus, in a preferred embodiment, a selected DNA sequence including sequences found in the wild-type von Willebrand gene, e.g., the six cytosines at positions 2679-2684 in exon 18 of the von Willebrand gene, can be used to target and correct the mutated von Willebrand gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the Xeroderma pigmentosum group G (XP-G) gene. Preferably, the mutation is a deletion of a single adenine in a stretch of three adenines at positions 19-21 of a 245 base-pair exon found in the XP-G gene. This deletion leads to xeroderma pigmentosum. Thus, in a preferred embodiment, a selected DNA including the wild-type sequence of the XP-G gene, e.g., three adenines at positions 19-21 of the 245 base-pair exon of the XP-G gene, can be used to target and correct the mutated XP-G gene.
In another preferred embodiment, the selected DNA sequence differs from the target DNA by more than one nucleotide, e.g., it differs from the target by a sufficient number of nucleotides such that the target, or the selected DNA sequence has an unpaired region, e.g., a loop-out region. Preferably, an agent which inactivates a mismatch repair protein such as Msh2, Msh6, Msh3, Mlh1, Pms2, Mlh3, Pms1, or combinations thereof, is also included in the composition, e.g., the agent can be included in the complex.
In a preferred embodiment, the selected DNA sequence has a flanking sequence such that it can integrate in a preselected relationship with a preselected element on a target DNA. For example, if the selected DNA is a regulatory sequence and the target DNA encodes a protein, the flanking sequence is such that it will integrate the regulatory element so that it functions to regulate expression of the protein encoding sequence. Flanking sequences which promote the selected integration can be used. The selected DNA sequence can have a flanking sequence such that it can be integrated 5xe2x80x2, 3xe2x80x2 or within, a selected target sequence, e.g., a gene or coding region in the target.
In a preferred embodiment, the selected DNA sequence includes a regulatory sequence, e.g., an exogenous regulatory sequence. In a preferred embodiment, the regulatory sequence includes one or more of: a promoter, an enhancer, an UAS, a scaffold-attachment region or a transcription factor-binding site. In a preferred embodiment, the regulatory sequence includes: a regulatory sequence from a metallothionein-I gene, e.g., the mouse metallothionein-I gene, a regulatory sequence from an SV-40 gene, a regulatory sequence from a cytomegalovirus gene, a regulatory sequence from a collagen gene, a regulatory sequence from an actin gene, a regulatory sequence from an immunoglobulin gene, a regulatory sequence from the HMG-CoA reductase gene, a regulatory sequence from xcex3 actin gene, a regulatory sequence from transcription activator YY1 gene, a regulatory sequence from fibronectin gene, or a regulatory sequence from the EF-1xcex1 gene.
In a preferred embodiment, the selected DNA sequence includes an exon. Preferably, the exogenous exon includes: a CAP site, the nucleotide sequence ATG, and/or encoding DNA in-frame with the targeted endogenous gene.
In a preferred embodiment, the selected DNA sequence includes a splice-donor site.
In a preferred embodiment, a composition which includes a selected DNA sequence having exogenous regulatory sequence can have a flanking sequence such that it is integrated into the target such that it functions to regulate expression of an endogenous sequence. The selected DNA can be integrated into the target upstream of the coding region of an endogenous gene or coding sequence in the target, or integrated into the target upstream of the endogenous regulatory sequence of an endogenous gene or coding sequence in the target. In another preferred embodiment, the selected DNA sequence can be integrated into the target such that the endogenous regulatory sequence of the endogenous gene is inactive, e.g., is wholly or partially deleted. The selected DNA sequence can be integrated into the target downstream of the endogenous gene or coding region, or integrated within an intron of an endogenous gene.
In a preferred embodiment, the selected DNA sequence includes a regulatory sequence, e.g., a regulatory sequence which differs in sequence from the regulatory sequence of the FSHxcex2 gene. Preferably, the selected DNA sequence is flanked by a targeting sequence, e.g., such targeting sequence is present at one or more, preferably both ends of the selected DNA sequence. In a preferred embodiment, the targeting sequence is homologous to a region 5xe2x80x2 of FSHxcex2 coding region (SEQ ID NO:1). In a preferred embodiment, the targeting sequence directs homologous recombination within the FSHxcex2 coding sequence, or upstream of the FSHxcex2 coding sequence. In a preferred embodiment, the targeting sequence includes at least 20, 30, 50, 100 or 1000 contiguous nucleotides from SEQ ID NO:2, which corresponds to nucleotides xe2x88x927454 to xe2x88x921417 of human FSHxcex2 sequence (numbering is relative to the translation start site), or SEQ ID NO:3, which corresponds to nucleotides xe2x88x92696 to xe2x88x92155 of human FSHxcex2 sequence.
In a preferred embodiment, the selected DNA sequence includes a regulatory sequence, e.g., a regulatory sequence which differs in sequence from the regulatory sequence of the IFNxcex12 gene. Preferably, the selected DNA sequence is flanked by a targeting sequence, e.g., such targeting sequence is present at one or more, preferably both ends of the selected DNA sequence. In a preferred embodiment, the targeting sequence is homologous to a region 5xe2x80x2 of IFNxcex12 coding region. In a preferred embodiment, the targeting sequence directs homologous recombination within a region upstream of the IFNxcex12 coding sequence. In a preferred embodiment, the targeting sequence includes at least 20, 30, 50, 100 or 1000 contiguous nucleotides from SEQ ID NO:4, which corresponds to nucleotides xe2x88x924074 to xe2x88x92511 of human IFNxcex12 sequence (numbering is relative to the translation start site). For example, it can include: at least 20, 30, 50, or 100 nucleotides from SEQ ID NO:7, which corresponds to nucleotides xe2x88x924074 to xe2x88x923796 of human IFNxcex12 sequence; at least 20, 30, or 50 nucleotides from SEQ ID NO:8, which corresponds to nucleotides xe2x88x92582 to xe2x88x92510 of human IFNxcex12 sequence; at least 20, 30, 50, 100, or 1000 nucleotides from SEQ ID NO:9, which corresponds to nucleotides xe2x88x923795 to xe2x88x92583 of human IFNxcex12 sequence.
In a preferred embodiment, the selected DNA sequence includes a regulatory sequence, e.g., a regulatory sequence which differs in sequence from the regulatory sequence of the GCSF gene. Preferably, the selected DNA sequence is flanked by a targeting sequence, e.g., such targeting sequence is present at one or more, preferably both ends of the selected DNA sequence. In a preferred embodiment, the targeting sequence is homologous to a region 5xe2x80x2 of GCSF coding region. In a preferred embodiment, the targeting sequence directs homologous recombination: within the GCSF coding sequence; upstream of the GCSF coding sequence. In a preferred embodiment, the targeting sequence includes at least 20, 30, 50, 100 or 1000 contiguous nucleotides from SEQ ID NO:5, which corresponds to nucleotides xe2x88x926,578 to 101 of human GCSF sequence (numbering is relative to the translation start site). For example, the target sequence can include 20, 30, 50, 100 or 1000 nucleotides from SEQ ID NO:6, which corresponds to nucleotides xe2x88x926,578 to xe2x88x92364 of the human GCSF gene (numbering is relative to the translation start site).
In another preferred embodiment, the DNA sequence includes a coding region, e.g., the DNA sequence encodes a protein. In a preferred embodiment, the coding region encodes: a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein or a transcription factor. In a preferred embodiment, the coding region encodes any of the following proteins: erythropoietin, calcitonin, growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, xcex12-interferon (IFNA2), xcex2-interferon, xcex3-interferon, nerve growth factors, FSHxcex2, TGF-xcex2, tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-xcex2, interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-granulocyte (GCSF), CSF-macrophage, CSF-granulocyte/macrophage, immunoglobulins, catalytic antibodies, protein kinase C, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNAse, xcex1-galactosidase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 antagonists, xcex11-antitrypsin, immune response modifiers, xcex2-glucoceramidase, xcex1-iduronidase, xcex1-L-iduronidase, glucosamine-N-sulfatase, xcex1-N-acetylglucosaminidase, acetylcoenzymeA:xcex1-glucosamine-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, xcex2-galactosidase, xcex2-glucuronidase, N-acetylgalactosamine-6-sulfatase, and soluble CD4.
In a preferred embodiment, the selected DNA sequence can have a flanking sequence such that when it is integrated into the target it is under the control of an endogenous regulatory element. The selected DNA can be integrated downstream of an endogenous regulatory sequence or upstream of a coding region of an endogenous gene and downstream of the endogenous regulatory sequence of the gene. In another preferred embodiment, the selected DNA can be integrated downstream of an endogenous regulatory sequence such that the coding region of the endogenous gene is inactive, e.g., is wholly or partially deleted.
In a preferred embodiment, the composition, e.g., the complex, is introduced into a cell. Preferably, the cell is a eukaryotic cell. In a preferred embodiment, the cell is of fungal, plant or animal origin, e.g., vertebrate origin. In a preferred embodiment, the cell is: a mammalian cell, e.g., a primary or secondary mammalian cell, e.g., a fibroblast, a hematopoietic stem cell, a myoblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a cell comprising a formed element of the blood, a muscle cell and precursors of these somatic cells; a transformed or immortalized cell line. Preferably, the cell is a human cell. Examples of immortalized human cell line useful in the present method include, but are not limited to: a Bowes Melanoma cell (ATCC Accession No. CRL 9607), a Daudi cell (ATCC Accession No. CCL 213), a HeLa cell and a derivative of a HeLa cell (ATCC Accession Nos. CCL2 CCL2.1, and CCL 2.2), a HL-60 cell (ATCC Accession No. CCL 240), a HT1080 cell (ATCC Accession No. CCL 121), a Jurkat cell (ATCC Accession No. TIB 152), a KB carcinoma cell (ATCC Accession No. CCL 17), a K-562 leukemia cell (ATCC Accession No. CCL 243), a MCF-7 breast cancer cell (ATCC Accession No. BTH 22), a MOLT-4 cell (ATCC Accession No. 1582), a Namalwa cell (ATCC Accession No. CRL 1432), a Rafji cell (ATCC Accession No. CCL 86), a RPMI 8226 cell (ATCC Accession No. CCL 155), a U-937 cell (ATCC Accession No. 1593), WI-28VA13 sub line 2R4 cells (ATCC Accession No. CLL 155), a CCRF-CEM cell (ATCC Accession No. CCL 119) and a 2780AD ovarian carcinoma cell (Van Der Blick et al., Cancer Res. 48:5927-5932, 1988), as well as heterohybridoma cells produced by fusion of human cells and cells of another species. In another embodiment, the immortalized cell line can be cell line other than a human cell line, e.g., a CHO cell line, a COS cell line.
In a preferred embodiment, the composition further includes an agent which inhibits a mismatch-repair protein, e.g., Msh2, Msh6, Msh3, Mlh1, Pms2, Mlh3, Pms1, or other mismatch repair proteins, or combinations thereof. Preferably, the agent is an agent which inhibits expression of a mismatch-repair protein, e.g., the agent is an antisense RNA. In a preferred embodiment, the agent is an antibody against a mismatch-repair protein. In a preferred embodiment, the antibody against the mismatch-repair protein is covalently or non-covalently linked to one or more components of the composition.
In another aspect, the invention features, a method of providing a protein. The method includes: providing a cell made by a method described herein, and allowing the cell to express the protein.
In a preferred embodiment: the method includes: providing a cell in which the following components have been introduced at a targeted site for alteration: (a) a double stranded DNA sequence which includes a selected DNA sequence; (b) an agent which enhances homologous recombination, e.g., a Rad52 protein or a functional fragment thereof; and (c) an agent which inhibits non-homologous end joining, e.g., a Ku inactivating agent; and allowing the cell to express the protein. Expression of the protein can occur, for example, by allowing expression of a protein encoded by the DNA, or by activating expression of the protein.
In a preferred embodiment, components (a), (b), and (c) are provided, e.g., introduced into the cell, such that, at the site of an interaction between the selected DNA sequence and the target DNA, the concentrations of the agent which enhances homologous recombination and of the agent which inhibits non-homologous end joining are sufficient that an alteration of the site, e.g., homologous recombination or gene correction, between the selected DNA sequence and the target DNA, occurs at a higher rate than would occur in the absence of the supplied agent which enhances homologous recombination and the agent which inhibits non-homologous end joining. The agent which inhibits non-homologous end joining is preferably provided locally.
In a preferred embodiment, components (a), (b), and (c) can be introduced together or separately. In addition, two of the components can be introduced together and the third can be introduced separately. For example, the DNA sequence and the agent which enhances homologous recombination, e.g., Rad52, can be introduced together or the DNA sequence and the agent which inhibits non-homologous end joining, e.g., a Ku inactivating agent, can be introduced together. In another preferred embodiment, the agent which enhances homologous recombination and the agent which inhibits non-homologous end joining can be introduced together.
Two, or preferably all, of the components can be provided as a complex. In a preferred embodiment, the method includes contacting the target DNA, e.g., by introducing into the cell, a complex which includes: (a) a double stranded DNA sequence which includes the selected DNA sequence; (b) an agent which enhances homologous recombination, e.g., a Rad52 protein or functional fragment thereof; and (c) an agent which inhibits non-homologous end joining, e.g., an agent which inactivates Ku.
In a preferred embodiment, one, or more, preferably all of the components, are provided by local delivery, e.g., microinjection, and are not expressed from the target genome or other nucleic acid. In a particularly preferred embodiment, the agent which inhibits non-homologous end joining, e.g., a Ku-inactivating agent such as an anti-Ku antibody, is provided by local delivery, e.g., microinjection, and is not expressed from the target genome or other nucleic acid.
In a preferred embodiment, the agent which inhibits non-homologous end joining is: an agent which inactivates hMre11, e.g., an anti-hMre11 antibody or a hMre11-binding oligomer or polymer; an agent which inactivates hRad50, e.g., an anti-hRad50 antibody or a hRad50-binding oligomer or polymer; an agent which inactivates Nbs1, e.g., an anti-Nbs1 antibody or a hNbs1-binding oligomer or polymer; an agent which inactivates human ligase 4 (hLig4), e.g., an anti-hLig4 antibody or a hLig4-binding oligomer or polymer; an agent which inactivates hXrcc4, e.g., an anti-hXrcc4 antibody or a hXrcc4-binding oligomer or polymer; an agent which inactivates a human homolog of Rap1, e.g., an antibody to a human homolog of Rap1 or an oligomer or polymer which binds a human homolog of Rap1; an agent which inactivates a human homolog of Sir2304, e.g., an antibody to a human homolog of Sir2304 or an oligomer or polymer which binds a human homolog of Sir2304; an agent which inactivates Ku, e.g., an anti-Ku antibody or a Ku-binding oligomer or polymer. Any of the agents which inhibit non-homologous end joining can be administered alone or can be administered in combination with one or more of the other agents which inhibit non-homologous end joining.
In a preferred embodiment, the DNA sequence is a linear DNA sequence. In a preferred embodiment, the linear DNA sequence can have one or more single stranded overhang(s).
In a preferred embodiment, the selected DNA sequence is flanked by a targeting sequence. The targeting sequence is homologous to the target, e.g., homologous to DNA adjacent to the site where the target DNA is to be altered or to the site where the selected DNA sequence is to be integrated. Such flanking sequence can be present at one or more, preferably both ends of the selected DNA sequence. If two flanking sequences are present one should be homologous with a first region of the target and the other should be homologous to a second region of the target.
In a preferred embodiment, the DNA sequence has one or more protruding single stranded end, e.g., one or both of the protruding ends are 3xe2x80x2 ends or 5xe2x80x2 ends.
In a preferred embodiment, the agent which enhances homologous recombination is: a Rad52 protein or a functional fragment thereof; a Rad51 protein or a functional fragment thereof; a Rad54 protein or a functional fragment thereof; or a combination thereof.
In a preferred embodiment, the agent which enhances homologous recombination is adhered to, e.g., coated on, the DNA sequence. In a preferred embodiment, the Rad52 protein or functional fragment thereof is adhered to, e.g., coated, on the selected DNA sequence.
In a preferred embodiment, the Rad52 protein or fragment thereof is human Rad52 (hRad52).
In a preferred embodiment, the anti-Ku antibody is: an anti-Ku70 antibody; an anti-Ku80 antibody. In a preferred embodiment, the anti-Ku antibody is: a humanized antibody; a human antibody; an antibody fragment, e.g., a Fab, Fabxe2x80x2, F(abxe2x80x2)2 or F(v) fragment.
In a preferred embodiment, at least one anti-Ku antibody is covalently linked to: the selected DNA sequence; the agent which enhances homologous recombination, e.g., the Rad52 protein or fragment thereof. In another preferred embodiment, at least one anti-Ku antibody is non-covalently linked to: the selected DNA sequence; the agent which enhances homologous recombination, e.g., the rad52 protein or fragment thereof.
In a preferred embodiment, the complex includes an anti-Ku70 antibody and an anti-Ku80 antibody provided, e.g., as components of a complex.
In a preferred embodiment, the cell is: a eukaryotic cell. In a preferred embodiment, the cell is of fungal, plant or animal origin, e.g., vertebrate origin. In a preferred embodiment, the cell is: a mammalian cell, e.g., a primary or secondary mammalian cell, e.g., a fibroblast, a hematopoietic stem cell, a myoblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a cell comprising a formed element of the blood, a muscle cell and precursors of these somatic cells; a transformed or immortalized cell line. Preferably, the cell is a human cell. Examples of immortalized human cell line useful in the present method include, but are not limited to: a Bowes Melanoma cell (ATCC Accession No. CRL 9607), a Daudi cell (ATCC Accession No. CCL 213), a HeLa cell and a derivative of a HeLa cell (ATCC Accession Nos. CCL2 CCL2.1, and CCL 2.2), a HL-60 cell (ATCC Accession No. CCL 240), a HT1080 cell (ATCC Accession No. CCL 121), a Jurkat cell (ATCC Accession No. TIB 152), a KB carcinoma cell (ATCC Accession No. CCL 17), a K-562 leukemia cell (ATCC Accession No. CCL 243), a MCF-7 breast cancer cell (ATCC Accession No. BTH 22), a MOLT-4 cell (ATCC Accession No. 1582), a Namalwa cell (ATCC Accession No. CRL 1432), a Rafji cell (ATCC Accession No. CCL 86), a RPMI 8226 cell (ATCC Accession No. CCL 155), a U-937 cell (ATCC Accession No. 1593), WI-28VA13 sub line 2R4 cells (ATCC Accession No. CLL 155), a CCRF-CEM cell (ATCC Accession No. CCL 119) and a 2780AD ovarian carcinoma cell (Van Der Blick et al., Cancer Res. 48:5927-5932, 1988), as well as heterohybridoma cells produced by fusion of human cells and cells of another species. In another embodiment, the immortalized cell line can be cell line other than a human cell line, e.g., a CHO cell line, a COS cell line.
In a preferred embodiment, the components, e.g., the components of a complex, are introduced into the cell by microinjection.
In a preferred embodiment, the method further includes introducing an agent which inhibits a mismatch-repair protein, e.g., Msh2, Msh6, Msh3, Mlh1, Pms2, Mlh3, Pms1, or other mismatch repair proteins or combinations thereof. Preferably, the agent is an agent which inhibits expression of a mismatch-repair protein, e.g., the agent is an antisense RNA. In a preferred embodiment, the agent is an antibody against a mismatch-repair protein. In a preferred embodiment, the antibody against the mismatch-repair protein is covalently or non-covalently linked to the complex.
In a preferred embodiment, the protein is expressed in vitro. In other preferred embodiments, the cell is provided in a subject, e.g., a human, and the protein is expressed in the subject. In a preferred embodiment, the protein is expressed in a subject and the cell is autologous, allogeneic or xenogeneic. Selected DNA can be introduced into a cell in vivo, or the cell can be removed from the subject, the selected DNA introduced ex vivo, and the cell returned to the subject.
In a preferred embodiment, the selected DNA sequence differs from the target DNA by less than 10, 8, 6, 5, 4, 3, 2, or by a single nucleotide, e.g., a substitution, or a deletion, or an insertion.
In a preferred embodiment, the target DNA includes a mutation, e.g., the target sequence differs from wild-type sequence by about 10, 8, 6, 5, 4, 3, 2 or by a single nucleotide. Preferably, the mutation is a point mutation, e.g., a mutation due to an insertion, deletion or a substitution.
In a preferred embodiment, the target DNA includes a mutation and the mutation is associated with, e.g., causes, contributes to, conditions or controls, a disease or a dysfunction. Preferably, the disease or dysfunction is: cystic fibrosis; sickle cell anemia; hemophilia A; hemophilia B; von Willebrand disease type 3; xeroderma pigmentosa; thalassaemias; Lesch-Nylan syndrome; protein C resistance; a lysosomal disease, e.g., Gaucher disease, Fabry disease, mucopolysaccharidosis (MPS) type 1 (Hurley-Scheie syndrome), MPS type II (Hunter syndrome), MPS type IIIA (Sanfilio A syndrome), MPS type IIIB (Sanfilio B syndrome), MPS type IIIC (Sanfilio C syndrome), MPS type IIID (Sanfilio D syndrome), MPS type IVA (Morquio A syndrome), MPS type IVB (Morquio B syndrome), MPS type VI (Maroteaux-Larry syndrome), MPS type VII (Sly syndrome).
In a preferred embodiment, the target DNA includes a mutation and the selected DNA sequence includes a normal wild-type sequence which can correct the mutation.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the cystic fibrosis transmembrane regulator (CFTR) gene. Preferably, the mutation is one which alters the amino acid at codon 508 of the CFTR protein-coding region, e.g., the mutation is a 3 base pair in-frame deletion which eliminates a phenylalanine at codon 508 of the CFTR protein. This deletion of phenylalanine-508 in the CFTR protein is found in a high percentage of subjects having cystic fibrosis. Thus, in a preferred embodiment, a selected DNA sequence including sequence encoding phenylalanine-508 as found in the wild-type CFTR gene can be used to target and correct the mutated CFTR gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the human xcex2-globin gene. Preferably, the mutation is one which alters the amino acid at the sixth codon of the xcex2-globin gene, e.g., the mutation is an A to T substitution in the sixth codon of the xcex2-globin gene. This mutation leads to a change from glutamic acid to valine in the xcex2-globin protein which is found in subjects having sickle cell anemia. Thus, in a preferred embodiment, a selected DNA which encodes a wild-type amino acid residue at codon 6, e.g., a selected DNA sequence including an A as found within the sixth codon of wild-type xcex2-globin gene, can be used to target and correct the mutated xcex2-globin gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the Factor VIII gene. For example, a mutation can be in exon 23, 24, and/or exon 25 of the Factor VIII gene. Preferably, the mutation is one which alters the amino acid at codon 2209 of the coding region of the Factor VIII protein coding region, e.g., the mutation is a G to A substitution in exon 24 of the Factor VIII gene which leads to a change from an arginine to a glutamine at amino acid 2209 of Factor VIII. Preferably, the mutation is one which alters the amino acid at codon 2229 of the coding region of the Factor VIII protein coding region, e.g., the mutation is a G to T substitution in exon 25 of the Factor VIII gene which leads to a change from a tryptophan to a cysteine at amino acid 2229 of Factor VIII. These mutations have been associated with moderate to severe hemophilia A. Thus, in a preferred embodiment, a selected DNA sequence including either DNA which encodes a wild-type amino acid at codon 2209 of the coding region of Factor VIII gene, or DNA which encodes a wild-type amino acid at codon 2229 of the coding region of the Factor VIII gene, or both, can be used to target and correct the mutated Factor VIII gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the Factor IX gene. For example, in subjects having hemophilia B, most of the mutations are point mutations in the Factor IX gene. Thus, in a preferred embodiment, the selected DNA sequence can include one or more nucleotides having at least one nucleotide from the wild-type Factor IX gene, to target and correct one or more of the point mutations in the Factor IX gene associated with hemophilia B.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the von Willebrand factor gene. Preferably, the mutation is a single cytosine deletion in a stretch of six cytosines at positions 2679-2684 in exon 18 of the von Willebrand gene. This mutation is found in a significant percentage of subjects having von Willebrand disease type 3. Other mutations, e.g., point mutations, associated with von Willebrand disease type 3 can also be altered as described herein. Thus, in a preferred embodiment, a selected DNA sequence including sequences found in the wild-type von Willebrand gene, e.g., the six cytosines at positions 2679-2684 in exon 18 of the von Willebrand gene, can be used to target and correct the mutated von Willebrand gene.
In a preferred embodiment, the target DNA includes a mutation and the mutation is in the Xeroderma pigmentosum group G (XP-G) gene. Preferably, the mutation is a deletion of a single adenine in a stretch of adenines at positions 19-21 of a 245 base-pair exon found in the XP-G gene. This deletion leads to xeroderma pigmentosum. Thus, in a preferred embodiment, a selected DNA including the wild-type sequence of XP-G gene, e.g., three adenines at positions 19-21 at the 245 base-pair exon of the XP-G gene, can be used to target and correct the mutated XP-G gene.
In another preferred embodiment, the alteration includes homologous recombination between the selected DNA sequence and the target DNA, e.g., a chromosome.
In preferred embodiment, the selected DNA sequence differs from the target DNA by more than one nucleotide, e.g., it differs from the target by a sufficient number of nucleotides such that the target, or the selected DNA sequence has an unpaired region, e.g., a loop-out region. In such an application, Msh2, Msh6, Msh3, Mlh1, Pms2, Mlh3, Pms1, or combinations thereof, can also be provided, e.g., as part of a complex.
In a preferred embodiment, the alteration includes integration of the selected sequence into the target DNA and the selected DNA is integrated such that it is in a preselected relationship with a preselected element on the target, e.g., if one is a regulatory element and the other is a sequence which encodes a protein, the regulatory element functions to control expression of the protein encoding sequence. Flanking sequences which promote the selected integration can be used. The selected DNA sequence can be integrated 5xe2x80x2, 3xe2x80x2, or within, a selected target sequence, e.g., a gene or coding sequence.
In a preferred embodiment, the alteration includes integration of the selected DNA sequence and the selected DNA sequence is a regulatory sequence, e.g., an exogenous regulatory sequence. In a preferred embodiment, the regulatory sequence includes one or more of: a promoter, an enhancer, an UAS, a scaffold-attachment region or a transcription factor-binding site. In a preferred embodiment, the regulatory sequence includes: a regulatory sequence from metallothionein-I gene, e.g., the mouse metallothionein gene, a regulatory sequence from an SV-40 gene, a regulatory sequence from a cytomegalovirus gene, a regulatory sequence from a collagen gene, a regulatory sequence from an actin gene, a regulatory sequence from an immunoglobulin gene, a regulatory sequence from the HMG-CoA reductase gene, a regulatory sequence from xcex3 actin gene, a regulatory sequence from transcription activator YY1 gene, a regulatory sequence from fibronectin gene, or a regulatory sequence from the EF-1xcex1 gene.
In a preferred embodiment, the selected DNA sequence includes an exon. Preferably, the exogenous exon includes: a CAP site, the nucleotide sequence ATG, and/or encoding DNA in-frame with the targeted endogenous gene.
In a preferred embodiment, the selected DNA sequence includes a splice-donor site.
In a preferred embodiment, the selected DNA sequence includes an exogenous regulatory sequence which when integrated into the target functions to regulate expression of an endogenous gene. The selected DNA can be integrated upstream of the coding region of an endogenous gene in the target or upstream of the endogenous regulatory sequence of an endogenous gene or coding region in the target. In another preferred embodiment, the selected DNA can be integrated downstream of an endogenous gene or coding region or within an intron or endogenous gene. In another preferred embodiment, the endogenous regulatory sequence of the endogenous gene is inactive, e.g., is wholly or partially deleted.
In a preferred embodiment, the selected DNA sequence is upstream of the endogenous gene and is linked to the second exon of the endogenous gene.
In a preferred embodiment, the endogenous gene encodes: a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein or a transcription factor. In a preferred embodiment, the endogenous gene encodes any of the following proteins: erythropoietin, calcitonin, growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, xcex22-interferon (IFNA2), xcex2-interferon, xcex3-interferon, nerve growth factors, FSHxcex2, TGF-xcex2, tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-xcex2, interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-granulocyte (GCSF), CSF-macrophage, CSF-granulocyte/macrophage, immunoglobulins, catalytic antibodies, protein kinase C, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNAse, xcex1-galactosidase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 antagonists, xcex1-I-antitrypsin, immune response modifiers, xcex2-glucoceramidase, xcex1-iduronidase, xcex1-L-iduronidase, glucosamine-N-sulfatase, xcex1-N-acetylglucosaminidase, acetylcoenzymeA:xcex1-glucosamine-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, xcex2-galactosidase, xcex2-glucuronidase, N-acetylgalactosamine-6-sulfatase, and soluble CD4.
In a preferred embodiment, the endogenous gene encodes follicle stimulating hormone xcex2(FSHxcex2) and the selected DNA sequence includes a regulatory sequence, e.g., a regulatory sequence which differs in sequence from the regulatory sequence of the FSHxcex2 gene. Preferably, the selected DNA sequence is flanked by a targeting sequence, e.g., such targeting sequence is present at one or more, preferably both ends of the selected DNA sequence. In a preferred embodiment, the targeting sequence is homologous to a region 5xe2x80x2 of FSHxcex2 coding region (SEQ ID NO:1). In a preferred embodiment, the targeting sequence directs homologous recombination within the FSHxcex2 coding sequence or upstream of the FSHxcex2, coding sequence. In a preferred embodiment, the targeting sequence includes at least 20, 30, 50, 100 or 1000 contiguous nucleotides from SEQ ID NO:2, which corresponds to nucleotides xe2x88x927454 to xe2x88x921417 of human FSHxcex2 sequence (numbering is relative to the translation start site), or SEQ ID NO:3, which corresponds to nucleotides xe2x88x92696 to xe2x88x92155 of human FSHxcex2 sequence.
In a preferred embodiment, the endogenous gene encodes interferon xcex12 (IFNxcex12) and the selected DNA sequence includes a regulatory sequence, e.g., a regulatory sequence which differs in sequence from the regulatory sequence of the IFNxcex12 gene. Preferably, the selected DNA sequence is flanked by a targeting sequence, e.g., such targeting sequence is present at one or more, preferably both ends of the selected DNA sequence. In a preferred embodiment, the targeting sequence is homologous to a region 5xe2x80x2 of IFNxcex12 coding region. In a preferred embodiment, the targeting sequence directs homologous recombination within a region upstream of the IFNxcex12 coding sequence. In a preferred embodiment, the targeting sequence includes at least 20, 30, 50, 100 or 1000 contiguous nucleotides from SEQ ID NO:4, which corresponds to nucleotides xe2x88x924074 to xe2x88x92511 of human IFNxcex12 sequence (numbering is relative to the translation start site). For example, it can include: at least 20, 30, 50, or 100 nucleotides from SEQ ID NO:7, which corresponds to nucleotides xe2x88x924074 to xe2x88x923796 of human IFNxcex12 sequence; at least 20, 30, or 50 nucleotides from SEQ ID NO:8, which corresponds to nucleotides xe2x88x92582 to xe2x88x92510 of human IFNxcex12 sequence; at least 20, 30, 50, 100, or 1000 nucleotides from SEQ ID NO:9, which corresponds to nucleotides xe2x88x923795 to xe2x88x92583 of human IFNxcex12 sequence.
In a preferred embodiment, the endogenous gene encodes granulocyte colony stimulating factor (GCSF) and the selected DNA sequence includes a regulatory sequence, e.g., a regulatory sequence which differs in sequence from the regulatory sequence of the GCSF gene. Preferably, the selected DNA sequence is flanked by a targeting sequence, e.g., such targeting sequence is present at one or more, preferably both ends of the selected DNA sequence. In a preferred embodiment, the targeting sequence is homologous to a region 5xe2x80x2 of GCSF coding region. In a preferred embodiment, the targeting sequence directs homologous recombination within the GCSF coding sequence or upstream of the GCSF coding sequence. In a preferred embodiment, the targeting sequence includes at least 20, 30, 50, 100 or 1000 contiguous nucleotides from SEQ ID NO:5, which corresponds to nucleotides xe2x88x926,578 to 101 of human GCSF sequence (numbering is relative to the translation start site). For example, the target sequence can include 20, 30, 50, 100 or 1000 nucleotides from SEQ ID NO:6, which corresponds to nucleotides xe2x88x926,578 to xe2x88x92364 of the human GCSF gene (numbering is relative to the translation start site).
In another preferred embodiment, the DNA sequence includes a coding region, e.g., the DNA sequence encodes a protein. In a preferred embodiment, the coding region encodes: a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein or a transcription factor. In a preferred embodiment, the coding region encodes any of the following proteins: erythropoietin, calcitonin, growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, xcex2-interferon, xcex3-interferon, nerve growth factors, FSHxcex2, TGF-xcex2, tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-xcex2, interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-granulocyte, CSF-macrophage, CSF-granulocyte/macrophage, immunoglobulins, catalytic antibodies, protein kinase C, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNAse, xcex1-galactosidase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 antagonists, xcex1-1-antitrypsin, immune response modifiers, xcex2-glucoceramidase, xcex1-iduronidase, xcex1-L-iduronidase, glucosamine-N-sulfatase, xcex1-N-acetylglucosaminidase, acetylcoenzymeA:xcex1-glucosamine-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, xcex2-galactosidase, xcex2-glucuronidase, N-acetylgalactosamine-6-sulfatase, and soluble CD4.
In a preferred embodiment, the selected DNA sequence can be integrated into the target downstream of an endogenous regulatory sequence or upstream of a coding region of an endogenous gene and downstream of the endogenous regulatory sequence of the gene. In another preferred embodiment, the selected DNA sequence can be integrated downstream of an endogenous regulatory sequence such that the coding region of the endogenous gene is inactive, e.g., is deleted.
In another aspect, the invention features, a cell made by any of the methods described herein.
In another aspect, the invention features a method of altering expression of a protein coding sequence of a gene in a cell, by any of the methods described herein.
In a preferred embodiment, the method includes introducing a complex described herein having a DNA sequence which includes a regulatory sequence into the cell; maintaining the cell under conditions which permit alteration of a targeted genomic sequence to produce a homologously recombinant cell; and maintaining the homologously recombinant cell under conditions which permit expression of the protein coding sequence of the gene under control of the regulatory sequence.
Maintaining the homologously recombinant cell under conditions which permit expression of the protein coding sequence of the gene under control of the regulatory sequence, thereby altering expression of the protein coding sequence of the gene.
The term xe2x80x9chomologousxe2x80x9d as used herein, refers to a targeting sequence that is identical to or sufficiently similar to a target site, e.g., a chromosomal DNA target site, so that the targeting sequence and the target site can undergo homologous recombination. A small percentage of base pair mismatches is acceptable, as long as homologous recombination can occur at a useful frequency.
As used herein, the term xe2x80x9cwild-typexe2x80x9d refers to a sequence which is not associated with, e.g., causes, contributes to, conditions or controls, a disease or dysfunction.
As used herein, a xe2x80x9ccomplexxe2x80x9d refers to a stable association in which the components are coupled by covalent or non-covalent bonds.